Cassette for electrophoretic gels

ABSTRACT

An improved cassette for use in the formation of an electrophoretic gel comprises two plates with substantially planar walls having two sides and two ends so arranged in a side by side spaced apart array to form a gel receiving space between them. A plurality of dividing ribs on one or each of the walls extend from a first end of the wall or walls substantially parallel to at least one of the sides thereof to a rib base end. The ribs extend into the space so as to subdivide at least one end of the space into a plurality of substantially parallel wells. A plurality of holes extend through at least one of the walls of the cassette located at or adjacent the base of the dividing rib and aligned with the rib. The arrangement of holes allow sufficient current flow to replace that lost due to the dividing ribs. The structure retains the advantage of having solid dividing walls which are considerably more resilient than dividing walls made from fingers of gel even when reinforced with pegs or projections and yet by virtue of the provision of a hole at the base of the ribs allows sufficient current flow at the base of each rib that the current flow through the space is substantially uniform, thus preventing spreading of the bands as they form in the body of the gel.

This application is a continuation under 35 U.S.C. 120 of U.S.application Ser. No. 09/543,855, filed on Apr. 5, 2000, now U.S. Pat.No. 6,413,402 B1.

FIELD OF THE INVENTION

The present invention relates to a cassette for use in the formation ofan electrophoretic gel and more particularly to such a cassette whichincludes means to assist in separating and defining individual samplereceiving wells at one end of the electrophoretic gel.

BACKGROUND ART

Electrophoretic gels, usually comprising hydrogels such as agarose orpolyacrylamide are used for the separation of nucleic acids, proteinsand other macromolecular compounds. The sample to be separated is placedat one end of the gel and a direct electric field is applied between theends of the gel causing the components of the sample to migrate throughthe gel at rates dependent upon their molecular size and charge.

A mixture of components to be separated is normally introduced into oneof a number of small wells formed in an upper edge of the gel before theelectric current is applied. It is usual to run a number of suchmixtures simultaneously on an electrophoretic gel in a side by sidearrangement. For this purpose one mixture is placed in each of a seriesof wells formed in the upper edge of the gel.

In the past electrophoretic gels were formed by juxtaposing a pair ofglass plates in a slightly spaced apart side-by-side relationship andfilling the space therebetween with a liquid which can set and form anelectrophoretic gel. The side edges of the space between the glassplates were typically sealed with adhesive tape or a similar material,and when the gel is poured a comb was placed in the upper end of thespace between the glass plates. After the gel had set the comb would bewithdrawn leaving a series of spaced apart wells defined in the top ofthe gel, each well having been defined by one tooth of the comb. Atongue of gel remains between the glass plates separating the pair ofadjacent wells.

The gel is immersed in a buffer solution and electrodes above and belowthe gel cause a direct electric current to flow through the gel. Thecomponents in the mixtures travel through the gel from the top to thebottom of the gel at different rates depending on their size and chargeand separate out into bands.

In more recent years it has been proposed to preform electrophoreticgels in cassettes formed of synthetic plastics materials. The side wallsof the cassette are formed with integral means to connect them togetheralong the sides of the cassette. Again when the gel is poured a comb isplaced in the upper end of the space between the walls of the cassettefor the formation of the spaced apart wells. However, this arrangementhas a disadvantage that upon withdrawal of the comb the fingers of thegel may, with time, show an increased tendency to break away from theremainder of the gel. This results in poorly defined wells.Alternatively, if the tongues of gels are left intact upon withdrawal ofthe comb they may not firmly adhere to the plastic side wall of thecassette. This has resulted that the tongues may fall over side waysoccluding an adjacent well.

One attempt to overcome this problem is described in U.S. Pat. No.5,288,465, where ribs are provided in the cassette walls to define wellsat one end of the cassette. Whilst this arrangement provides stablewells suitable to hold the sample material, this arrangement has adisadvantage that the solid walls of the wells interfere with the smoothflow of electric current through the electrophoretic gel: because of theribs, the current is initially confined and then spreads out which has aresult that as the run proceeds bands from adjoining wells tend tospread out and merge with one another.

International Patent Application No WO 97/04307 addresses the problem ofcurrent interference by replacing the ribs by a plurality of smallprojections or pegs. These projections or pegs extend into the gel tosupport the gel fingers. The projections support the gel whilst allowingsubstantially parallel flow of the current to establish before thecurrent meets the samples positioned in the bottom of the wells. Thisparallel current flow is necessary to maintain the separation betweenthe bands/samples from the different wells and prevent the samples forspreading outwards.

However, the disadvantage of the cassette shown in WO 97/04307 is thatas the combs are withdrawn, the tops of the gel fingers tend to breakaway from the remainder of the gel finger.

The present invention is directed to alternative arrangements addressingthe problems associated with spreading current and also the integrity ofthe walls of the sample wells.

DISCLOSURE OF THE INVENTION

According to a first aspect of the present invention there is providedan improved cassette for use in the formation of an electrophoretic gelcomprising two plates with substantially planar walls having two sidesand two ends so arranged in a side by side spaced apart array to form agel receiving space between them, a plurality of dividing ribs on one oreach of the walls extending from a first end of the wall or wallssubstantially parallel to at least one of the sides thereof to a ribbase end and being adapted to extend into the space so as to subdivideat least one end of the space into a plurality of substantially parallelwells, the improvement consisting of a plurality of holes extendingthrough at least one of the walls of the cassette located at or adjacentthe base of the dividing rib and aligned with the rib, the arrangementbeing such that the holes allow sufficient current flow to replace thatlost due to the dividing ribs.

The present invention retains the advantage of having solid dividingwalls which are considerably more resilient than dividing walls madefrom fingers of gel even when reinforced with pegs or projections, andyet by virtue of the provision of a hole at the base of the ribs allowssufficient current flow at the base of each rib that the current flowthrough the space is substantially uniform, thus preventing spreading ofthe bands as they form in the body of the gel.

The size of the gap between the sample bands can be controlled byvarying the size of the hole in the wall of the cassette. A larger holecreates a greater current flow and provides a wider gap between thesamples. The size of the hole will depend on the thickness of the riband the size of the well. The holes may vary in size between about 2 mmdiameter to 0.5 mm diameter, with a 1 mm diameter hole being typical.

It is a preferred feature that the ribs are tapered outwardly from thefirst end to their base end so that the upper part of the well is widerthan the lower part of the well. This makes the wells easier to loadwith samples.

It is preferred that the sides of the holes are chamfered or tapered sothat during operation the chamfered hole will fill with buffer and nottrap air bubbles.

In gel cassettes of the type described above and in the introduction,the concentration of the gels is graduated. The cassettes are typicallyfilled from below which means that the gel at the top of the cassettewhich is the least dense, is the least concentrated. Ironically, incassettes of the type where gel fingers form, the walls of the well isalso the part where the gel needs to be strongest i.e. the mostconcentrated gel. Thus, there is a requirement for the concentration ofacrylamide in such cassettes to be high to ensure a high enough gelconcentration for forming the fingers. However, in the present inventionbecause the walls of the well are plastic and not gel, the upper zone ofthe gel can be a much lower concentration say around 2.5% as opposed to5% which allows the separation of a much higher molecular weightproteins having a molecular weight of around 800,000 to 1,000,000.

A second aspect of the present invention provides an improved cassettecontaining an electrophoretic gel comprising two plates withsubstantially planar walls having two sides and two ends so arranged ina side by side spaced apart array to form a gel receiving space betweenthem, a plurality of dividing ribs on one or each of the wall membersextending from the first end of the member or members substantiallyparallel to at least one of the sides thereof and adapted to extend intothe space so as to subdivide at least one end of the space into aplurality of substantially parallel wells having bases, the improvementcomprising that the part of the well walls formed from plastic ribmaterial does not extend to the base of the well and that the lower partof the well wall is formed from a finger of gel.

In one preferred embodiment, the finger of gel may be supported by oneor more projections.

The solid upper rib segment of the well wall resists stresses placed onthe gel fingers and maintains the integrity of the wells. It preventsdamage to the lower gel fingers defining the lower part of the wellwalls when the comb is withdrawn or when samples are loaded into thewells. Surprisingly the gel base of the wall allows the re-establishmentof parallel current flow through the gel before the well base is reachedby the current maintaining the separation between the bands ofseparating molecules in the body of the gel.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described, by way of example only, and withreference to the accompanying drawings in which:

FIG. 1 is a schematic front view of a cassette illustrating a firstembodiment of the present invention:

FIGS. 2 a and 2 b are schematic side views illustrating current flowthrough the cassette shown in FIG. 1;

FIG. 3 is a side view of a variant of the embodiment shown in FIG. 1;

FIG. 4 shows a further variant of the embodiment shown in FIG. 1; and

FIG. 5 shows a second embodiment of the present invention alsoillustrating separation between bands of molecules separated byelectrophoresis.

BRIEF DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Referring to the drawings FIG. 1 shows a first electrophoresis cassette10 which may be formed in two parts by injection moulding or by othermethods from a suitable synthetic plastic material.

One part forms a first side wall 11 of the cassette which is banded oneach side by a connecting means comprising a pair of spaced apart ridgesnot shown.

The other part forms a second side wall 12 of the cassette which is alsobanded on each side by a pair of spaced ridges, not shown. The ridgesinterdigitate to form a seal along each side of the cassette to preventleakage therethrough of the gel forming liquid during setting of the gelor flow of an electrical current during electrophoresis.

The ridges are of a sufficient thickness that a gel receiving space 13is defined between the side walls which lie in substantially parallelplanes. Such a construction as described above is already well known inthe prior art see, for example, WO 97/04307 the contents of which areincorporated herein by reference.

The inside surface of the side wall 11 is formed with a plurality ofinwardly directed parallel. spaced apart ribs 14 which extend into thegel receiving space 13.

The ribs 14 may however originate from either or both of walls 11 and12. Each rib has a length which defines the depth of the well, and athickness which spans the gel receiving space 13.

The ribs 14 extend from a first, upper end 11 a of side wall 11 to abase end 14 b which is located at the base of the well.

In an alternative embodiment, the ribs may be thicker than the gelreceiving space 13 and they may mate with corresponding recessesprovided in the opposite wall 12.

At the base of each rib distal from the upper end 11 a of the side wall11 there is a hole 15 which extends through the wall 11 and is typicallyaround 1 mm diameter.

To form a gel in the cassette 10 a comb with a plurality of spaced apartteeth is inserted into the upper end of the cassette. The teeth aredimensioned so that they interdigitate with the ribs 14 but are at alltimes closely spaced therefrom. After the gel is formed in the space 13the cassette can be used.

When the comb is withdrawn, there will be a plurality of wells formed inthe gel separated by the ribs 14.

In use the cassette is inserted in a buffer solution and current ispassed through the gel via an electrode located above the upper end ofthe cassette and an electrode located below the lower of end of thecassette. Current flows in two paths, the first path being through thewell illustrated by arrow (A) shown in FIG. 2 a and the second path (B)through the hole 15 thence through the gel to the base of the cassette.

It is that additional flow of current through the hole 15 that preventsthe current passing through the wells from spreading out andintermingling the bands.

The separation of the samples into bands of molecules is illustrated inFIG. 5, albeit in the context of a definite embodiment of the invention.The gap between bands can be varied by varying the size of the hole 15.

FIG. 3 shows a variant in which the side 16 of the hole 15 are taperedoutwardly or chamfered to allow buffer to fill the hole more easily andto prevent air bubbles becoming trapped in the hole 15. An air bubblewould inhibit the passage of electric current through the hole defeatingthe object of the invention.

FIG. 4 shows a further variant on the invention in which the ribs 14 aretapered being wider at their base portion adjacent the hole 15 andnarrower at the top portion adjacent the upper edge of side wall 11.This makes the upper part of the wells wider and makes loading the wellseasier.

FIG. 5 shows a further embodiment of the present invention in which theupper part of the walls of the dividing well are comprised of plasticribs 17 and the lower part of the walls comprise a gel finger 18 whichmay be stabilised with one or more pegs 19 such as described in WO97/04307.

The solid upper part of the rib resists stresses placed on the fingersand maintains integrity of the walls, particularly when the comb isbeing withdrawn or when samples are being loaded into the wells. Becausethe lower part of the well walls comprises a gel finger, this allows thecurrent to spread out sufficiently before it reaches the samples, forseparation to occur in even bands as illustrated in FIG. 4.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

1. A cassette for use in the formation of an electrophoretic gelcomprising two plates with substantially planar walls having two sidesand two ends so arranged in a side by side spaced apart array to form agel receiving space between the walls, a plurality of dividing ribs onone or each of the walls extending from a first end of the wall or wallssubstantially parallel to at least one of the sides thereof to a ribbase end and being adapted to extend into the space so as to subdivideat least one end of the space into a plurality of substantially parallelwells, the improvement consisting of a plurality of holes extendingthrough at least one of the walls of the cassette located at or adjacentthe rib base end of the dividing ribs and aligned with the dividingribs, the arrangement being such that the holes allow additional currentflow to replace current flow lost due to the dividing ribs.
 2. Thecassette as set forth in claim 1, wherein a diameter of the plurality ofholes is 1 mm.
 3. The cassette as set forth in claim 1, wherein adiameter of the plurality of holes is less than 1 mm.
 4. The cassette asset forth in claim 1, wherein the plurality of holes vary in sizebetween about 0.5 mm diameter to about 2 mm diameter.
 5. The cassette asset forth in claim 1, wherein the plurality of holes have a diameter ofabout 1 mm.
 6. The cassette as set forth in claim 1, wherein the ribsare tapered outwardly from an upper end of the ribs to a base end of theribs so that an upper part of the wells is wider than a lower part ofthe wells.
 7. The cassette as set forth in claim 1, wherein sides of theholes are chamfered or tapered.
 8. An improved cassette containing anelectrophoretic gel comprising two plates with substantially planarwalls having two sides and two ends so arranged in a side by side spacedapart array to form a gel receiving space between the walls, a pluralityof dividing ribs on one or each of the walls extending from a first endof the wall or walls substantially parallel to at least one of the sidesthereof and adapted to extend into the space so as to subdivide at leastone end of the space into a plurality of substantially parallel wellshaving bases, the improvement comprising that a part of the well wallsformed from plastic rib material does not extend to the respective baseof each of the wells and that a lower part of the well walls is formedfrom a finger of gel.
 9. The cassette as set forth in claim 8, whereinthe finger of gel is supported by one or more projections.
 10. Acassette for use in the formation of an electrophoretic gel comprisingtwo plates with walls having two sides and two ends so arranged in aside by side spaced apart array to form a gel receiving space betweenthe walls, at least one dividing rib on at least one of the wallsextending from a first end of the at least one of the walls to a ribbase end and being adapted to extend into the space so as to subdivideat least one end of the space into at least one well, the improvementconsisting of at least one hole extending through at least one of thewalls of the cassette and associated with the rib base end of the atleast one dividing rib, the arrangement being such that the at least onehole allows additional current flow to replace current flow lost due tothe at least one dividing rib.
 11. The cassette as set forth in claim10, wherein the at least one hole is substantially aligned with the atleast one dividing rib.
 12. The cassette as set forth in claim 10,wherein a diameter of the at least one hole is 1 mm.
 13. The cassette asset forth in claim 10, wherein a diameter of the at least one hole isless than 1 mm.
 14. The cassette as set forth in claim 10, wherein theat least one hole varies in size between about 0.5 mm diameter to about2 mm diameter.
 15. The cassette as set forth in claim 10, wherein the atleast one hole has a diameter of about 1 mm.
 16. The cassette of claim10, wherein the ribs are tapered outwardly from an upper end of the ribsto a base end of the ribs so that an upper part of the wells is widerthan a lower part of the wells.
 17. The cassette of claim 10, whereinsides of the at least one hole are chamfered or tapered.
 18. An improvedcassette containing an electrophoretic gel comprising two plates withwalls having two sides and two ends so arranged in a side by side spacedapart array to form a gel receiving space between the walls, at leastone dividing rib on at least one of the walls extending from a first endof the at least one of the walls and adapted to extend into the space soas to subdivide at least one end of the space into at least one wellhaving a base, the improvement comprising that a part of the at leastone dividing rib formed from plastic rib material does not extend to thebase of the at least one well and that a lower part of the at least onedividing rib is formed from a finger of gel.
 19. The cassette as setforth in claim 18, wherein the finger of gel is supported by one or moreprojections.
 20. A cassette, comprising: a first wall; a second wallcoupled to the first wall, the first wall and the second wall beingarranged so that a gel receiving space is formed between the first walland the second wall; at least one dividing rib on at least one of thefirst wall or the second wall, the at least one dividing rib extendingfrom a first end of the at least one of the first wall or the secondwall to a rib base end and being adapted to extend into the gelreceiving space to subdivide at least one end of the gel receiving spaceinto at least one well; and at least one hole extending through at leastone of the first wall or the second wall, the at least one hole beingassociated with the rib base end, wherein the at least one hole allowsadditional current flow to replace current flow lost due to the at leastone dividing rib.
 21. The cassette as set forth in claim 20, wherein theat least one hole is aligned with the at least one dividing rib.
 22. Acassette, comprising: a first wall; second wall coupled to the firstwall, the first wall and the second wall being arranged so that a gelreceiving space is formed between the first wall and the second wall;and at least one dividing rib on at least one of the first wall or thesecond wall, the at least one dividing rib extending from a first end ofthe at least one of the first wall or the second wall to a rib base endand being adapted to extend into the gel receiving space to subdivide atleast one end of the gel receiving space into at least one well, anupper part of the at least one dividing rib being formed from plasticrib material and a lower part of the at least one dividing rib beingformed from a finger of gel.